Platelet Rich Plasma (PRP) Matrix Grafts

Transcription

Platelet Rich Plasma
(PRP) Matrix Grafts
PRP application techniques in musculoskeletal medicine utilize the
concentrated healing components of a patient's own blood—reintroduced
into a specific site—to regenerate tissue and speed the healing process.
By David Crane, MD and Peter A.M. Everts, PhD
latelet Rich Plasma (PRP) grafting techniques are now
being utilized in musculoskeletal medicine with increasing frequency and effectiveness. Soft tissue injuries treated with PRP include tendonopathy, tendonosis, acute and chronic muscle strain, muscle fibrosis, ligamentous sprains, and joint
capsular laxity. PRP has also been utilized to treat intra-articular injuries. Examples include arthritis, arthrofibrosis, articular
cartilage defects, meniscal injury, and chronic synovitis or joint
inflammation.
Platelet Rich Plasma was first used in cardiac surgery by Ferrari et al. in 1987 as an autologous transfusion component after
an open heart operation to avoid homologous blood product
transfusion.1 It is now being utilized by musculoskeletal (MSK)
providers following the effective use in multiple specialties. PRP
has also been successfully used in various specialties such as maxillofacial, cosmetic, spine, orthopedic, podiatric and for general wound healing.2,3
MSK practitioners began using PRP for tendonosis and tendonitis in the early 1990s.4 PRP techniques have most commonly been applied by MSK practitioners previously trained in the
use of—and on the knowledge backbone of—prolotherapy. Although there is a paucity of well designed, randomized trials for
its use in MSK medicine, animal studies, case reports, and anecdotal evidence suggests that this technique will continue to develop as a way to regenerate tissue that has lost its inherent homeostasis and thereby relieve associated pain and dysfunction.
P
Standardizing the Nomenclature for PRP
The authors define a PRP Matrix Graft as follows:
A tissue graft incorporating autologous growth factors and/ or autologous undifferentiated cells in a cellular matrix whose design depends
on the receptor site and tissue of regeneration.
In reading the literature, different verbiage will arise, such as
1
platelet leukocyte gel, platelet rich plasma gel, platelet concentrate, blood plasma therapy, etc. When examining the literature,
one must evaluate whether concentrations of platelets, nucleated cells, growth factors, fibrin, and platelet activation is measured. These factors— along with skillful percutaneous injection
and surgical techniques—all contribute to the effectiveness of
therapy.5 Everts, on reviewing 28 human studies, found that
seven showed either no benefit or negative effects of PRP.3 However, when these studies were reviewed, many had very small
sample sizes (as few as three patients) and several had platelet
portions that had been activated prior to use via differing means.
Hopefully, in the near future, the nomenclature will benefit from
some form of standardization. It is the authors’ experience, however, that the wording of ‘graft’ is required in the nomenclature
for third party reimbursement reasons, as well as to accurately
describe how this modality is actually utilized at present in the
clinic and surgical settings.
For our purposes, we will consider PRP gel as PRP that is activated with either autologous thrombin and calcium, bovine
thrombin and calcium, or thrombin alone. Autologous PRP gel
stipulates the use of autologous thrombin. The author considers a PRP Matrix Graft to include gel or no gel. This must be
stipulated at the time of treatment. Again, the tissue of treatment will demand what matrix, if any, is added or utilized.
Constituents and Properties of an Effective Regenerative Graft
Normal tissue homeostasis is maintained in a prescribed physiologic manner. These stages will be reviewed from a hypothetical
time of injury through the healing phase to understand how to
maximize PRP graft matrix preparation. Platelets contain two
unique types of granules—the alpha-granules and dense granules.
Alpha-granules contain a variety of hemostatic proteins (coagulation proteins), as well as growth factors, cytokines,
Practical PAIN MANAGEMENT, January/February 2008
©2008 PPM Communications, Inc. Reprinted with permission.
chemokines (pro-inflammatory activation-inducible cytokines)
and other proteins such as adhesion proteins.6 Of primary interest to the clinician are the three adhesion molecules and seven
growth factors present in the alpha granule.7
Dense granules contain factors that promote platelet aggregation (ADP, calcium, serotonin). Cell activation of platelets
causes the discharge of granule contents. In other words,
platelets require activation in order to begin the cascade of
events that lead to collagen restoration and growth. This activation must occur at the tissue level (where the platelets aggregate and adhere to collagen at the site of grafting).5
A synopsis of the various growth factors in PRP, together with
their source and function, is presented in Table 1.
A PRP Matrix Graft is made in a clinical or operative setting
by using one of the several available table-top machines on the
market. Several authors offer reviews of available graft preparation centrifuges and their ability to concentrate growth factors.2,3,8 Each machine has a separate, disposable unit that concentrates platelets in a small amount of plasma. A thin layer of
platelets is found immediately above the leukocytes in the buffy
coat of centrifuged blood. When a concentrated platelet portion is made, the buffy coat containing elevated levels of leukocytes—along with concentrated platelets—are suspended in a
small amount of plasma for subsequent grafting. The clinician
hopes that the platelets are not activated and remain suspended until grafting and contact with thrombin or collagen occurs.
Necessary Stages of Healing
Normal platelet activation leads to three necessary stages of
healing: Inflammation, Proliferation, and Remodeling.9 The
cellular components involved in the three phases of healing are
FIGURE 1. The physiology of healing of the chronic wound. From: emedicine.com.15 Used with permission.
depicted in Figure 1. If any of these stages are incomplete—or
if they proceed unabated—tissue homeostasis is lost and pain
and loss of function may result. Most reviews on this topic focus
on only the growth factors contained within the alpha granule
of the platelet which is released upon platelet activation. It is
important to understand, however, that if the platelets aren’t
suspended with biologic levels of other constituents of plasma—
such as leukocytes, cytokines, and fibrin (the matrix)—the graft
is either not effective or less effective.3 If fibrin levels are too
high, or platelet activation occurs prior to collagen binding, the
graft is also inhibited. Other functions of platelet activation and
the subsequent cascade of events that occur include cytokine signaling, chemokine release, and mitogenesis.9
TABLE 1. SYNOPSIS OF GROWTH FACTORS PRESENT IN PRP
Growth Factor
Source
Function
Transforming Growth
Factor-beta,TGF-ß
Platelets, extracellular matrix
of bone, cartilage matrix,
activated TH1 cells and natural killer cells, macrophages/
monocytes and neutrophils
Stimulates undifferentiated mesenchymal cell proliferation; regulates
endothelial, fibroblastic and osteoblastic mitogenesis; regulates collagen synthesis and collagenase secretion; regulates mitogenic effects
of other growth factors; stimulates endothelial chemotaxis and angiogenesis; inhibits macrophage and lymphocyte proliferation
Basic Fibroblast
Growth Factor,bFGF
Platelets, macrophages,
mesenchymal cells, chondrocytes, osteoblasts
Promotes growth and differentiation of chondrocytes and osteoblasts;
mitogenetic for mesenchymal cells, chondrocytes and osteoblasts
Platelet Derived
Growth
Factor,PDGFa-b
Platelets, osteoblasts,
endothelial cells,
macrophages, monocytes,
smooth muscle cells
Mitogenetic for mesenchymal cells and osteoblasts; stimulates chemotaxis and mitogenesis in fibroblast/glial/smooth muscle cells; regulates
collagenase secretion and collagen synthesis; stimulates macrophage
and neutrophil chemotaxis
Epidermal Growth
Factor,EGF
Platelets, macrophages,
monocytes
Stimulates endothelial chemotaxis/angiogenesis; regulates collagenase
secretion; stimulates epithelial/mesenchymal mitogenesis
Vascular endothelial
growth factor,VEGF
Platelets, endothelial cells
Increases angiogenesis and vessel permeability, stimulates mitogenesis for endothelial cells
Connective tissue
growth factor,CTGF
Platelets through endocytosis from extracellular environment in bone marrow.
Promotes angiogenesis, cartilage regeneration, fibrosis and platelet
adhesion
TABLE 1. Synopsis of growth factors present in PRP From Peter A.M. Everts et al. Platelet-Rich Plasma and Platelet Gel: A Review3
2
900
hMSC Proliferation as a Function of
8-day Exposure to Platelet Released Concentrations
Scaffold
Quantity of hMSC’s
800
700
600
500
400
300
200
100
0
Platelet
Whole
Blood Poor Plasma
1.25x
2.5x
10x
5x
(x = increase above native levels)
FIGURE 2. Relationship of differing platelet concentrations and human
mesenchymal stem cell (hMSC) migration and proliferation. From:
Haynesworth, Stephen et al. Mitogenic Stimulation of Human Mesenchymal Stem Cells by Platelet Releasate.11 Used with permission.
Inflammatory Phase
During the inflammatory phase, the functions of activated
platelets include:
• Anti-microbial
• Adhesion
• Aggregation
• Clot retraction
• Pro-coagulation
• Cytokine signaling
• Chemokine release
• Growth factor release
There is now evidence to suggest that at certain concentrations, or dose response curves, platelet rich plasma grafts may
be anti-inflammatory or pro-inflammatory in certain tissues.10 A
dose response relationship exists to a currently unknown level
of PRP concentration and ensuing migration and proliferation
of progenitor stem cells at the tissue injury site11 (see Figure 2).
There is emerging evidence to suggest that PRP grafts in the
four- to six-fold range (106 platelets) have more anti-inflammatory mediators and effects and are clinically relevant and useful
for most situations. PRP grafts in the eight- to thirteen-fold
range may be pro-inflammatory in nature.10 Further elucidation
of this effect is required, however, as some studies showed beneficial effects of higher concentrations of PRP.12
Hesham El-Sharkawy et al. evaluated this effect in periodontal tissue. The conclusions were that PRP is a rich source of
growth factors and promoted significant changes in monocytemediated proinflammatory cytokine/chemokine release. LXA4
was increased in PRP, suggesting that PRP may suppress cytokine release, limit inflammation, and thereby promote tissue
regeneration.10
Weibrich et al. observed an advantageous effect with platelet
concentrations of approximately 106/µL. Further, they state
that higher concentrations might have a paradoxically inhibitory effect.13
Following the initial inflammatory phase, which typically lasts
for two to three days, fibroblasts enter the site and begin the
proliferative phase.9 Low pH and low oxygen levels stimulate
fibroblast proliferation in the injury site.14 Fibroblasts become
the most abundant cell by the seventh day. The fibroblasts are
3
Undifferentiated
Progenitor Cells
Signal Proteins and
Adhesion Molecules
FIGURE 3. Cell Proliferation Triangle.
then responsible for deposition of collagen and ground substance. This phase lasts from two to four weeks. As these are
primarily the deficient cells with chronic injury (lack of normal
collagen in extracellular matrix), this stage is mandatory for
MSK repair.
The Proliferative Phase
During the proliferative phase—peaking anywhere from day 5
to 15 and which can last for weeks—fibroblasts differentiate into
myofibroblasts and actin contracts to make the wound smaller.
Low pH and hypoxemia also stimulates neovascularization.
Neovessels begin to form at approximately day 5 to 7 and this
process proceeds until the neovessels disappear near completion of the remodeling phase.
The Remodeling Phase
During the remodeling phase, collagen matures and strengthens. Tissue repair starts when the production and break down
of collagen equalizes. This phase can last over one year. During
this period, type III collagen is replaced by type I collagen, reorganization occurs, and the blood neovessels disappear.9
Cell Proliferation Triangle
It has become apparent, then, that PRP grafts function via a
triad of interactions, known as the cell proliferation triangle16,17
(see Figure 3). Each element of this triangle must be present for
effective tissue repair and pain relief.
When preparing a graft for clinical use, the constituents of
each of these three must be considered—i.e. is there an inherent matrix to place the graft in, or will the graft be washed away
with motion, synovial fluid, or repeated graft compression or
distraction? Does the patient have an adequate response for inflammation and is there an adequate quantity of platelets to concentrate for progenitor cell mitogenesis and proliferation?
Biotensegrity—A Construct for Regeneration of Tissue
Biotensegrity refers to a dynamic construct of compressive and
tensional forces acting on, and through, multiple levels of organization to maintain or repair tissue homeostasis. Biotensegrity, then, is a repeated pattern of structural and functional architecture of all living tissue.19,20
The probable link though all levels of biotensegrity is the vascular endothelial system with its regenerative and neuroendocrine functions as subsequently described.
Endothelial cells line the lumen of all
blood vessels as a single squamous epithelial cell layer. They are derived from angioblasts and hemangioblasts. WeibelPalade bodies are specialized secretory
granules found in endothelial cells. These
vesicles store preformed hormones, cytokines, and growth factors; as well as enzymes, receptors, and adhesion molecules; which can be released and/or expressed on the cell surface without de
novo protein syntheses by regulated exocytosis in response to stimulation of cell
activation.6
Thus, the authors believe there is sufficient evidence to suggest that the vascular
endothelial system links all of the
biotensegrity levels together as the various
factors are at work up and down the scale.
Contraindications to the Use of PRP Matrix
Grafts3,21,22
Absolute Contraindications include:
• Platelet dysfunction syndrome
• Critical thrombocytopenia
• Hypofibrinogenemia
• Hemodynamic instability
• Septicemia
• Sensitivity to bovine thrombin (if
using bovine thrombin with calcium
to make platelet gel)
Relative Contraindications include:
• Consistent use (anti-inflammatory
use) of NSAID’s within 48 hours of
procedure
• Corticosteroid injection at treatment
site or systemic use of corticosteroids
within 2 weeks of graft procedure
• Recent fever or illness
• Rash at graft donor site or at receptor site
• Cancer — especially hematopoetic
or of bone
• Active history or history of
Pseudomonas,
Enterococcus
or
Klebsiella infection, as PRP has been
shown in one study to potentially
stimulate these pathogens.22
• HGB <10 g/dl
• Platelet count less than 105/µL
Risks Involved With the Use of PRP
Matrix Grafts
While there have been no reports of worsened pain or function following tissue
maturation that the authors could find,
few randomized, placebo controlled trials
exist regarding the utilization of these
grafts. In the primary author’s experience
of performing approximately 20 to 30
4
cases of percutaneous PRP Matrix Grafts
per week for the last two years, no patients
reported worsened pain or function. It is
felt by the authors—and often expressed
in the available literature— that this procedure technique is safe and effective.
Pain at the treatment site is common
for a short period following injection.
One of the primary author’s patients reported worsened pain for six months at a
treated lateral epicondyle. This subsequently resolved and has been absent for
over one year. This stresses the fact that
remodeling of the tissue is necessary to
see the effects of therapy. No tendon rupture or partial rupture was noted and the
authors can find no reports of tendon or
ligament rupture following PRP. In fact,
Olena Virchenko and Per Aspenberg
noted, in a rat achilles tendon transection
model, that one postoperative injection
resulted in increased strength after four
weeks. This effect was obliterated with the
use of botox at the site.18
Other risks that may occur at time of
injection include injury from pain-induced syncope. Indeed, the main complaint received from patients is the injection pain of the PRP. There is also the
risk of limb injury following the graft
procedure since local or regional anesthesia is used at the time of procedure.
The primary author had a patient who
stepped from a ladder about four hours
following an achilles and peroneal tendon injection, with subsequent inversion
and fracture of the ankle—most likely
due to proprioceptive and sensory loss
from anesthesia.
As with any percutaneous needle technique, there is a slight risk of puncturing
a hollow organ or infection, but this risk
is not expected to be above or below that
of other needle techniques employed in
clinical medicine. The accepted risk of introduction of infection with percutaneous
techniques has been reported as 1:50,000
injections. Since PRP is an autologous
preparation, the risk of introducing foreign material and the risk of transmissible infection or allergic reaction is effectively eliminated—although the entire
procedure must be carried out in sterile
conditions. PRP—with its initial inflammatory phase—is also bacteriocidal, particularly against Stapholococcus aureus
and Escherichia coli as shown by Bielecki et al.22 The temporary formation of
platelet and fibrin plugs at the wound site
has also been noted to prevent the entry
of microorganisms.3,22 However, PRP gel
seems to induce the in vitro growth of Ps.
aeruginosa, suggesting that it may cause
an exacerbation of infections with this organism. There was no activity against
Klebsiella pneumoniae or Enterococcus
faecalis.
Other considerations come into play if
the procedure is not performed with completely autologous preparations. PRP gel
techniques that rely upon the use of
bovine thrombin, which may contain contaminants like bovine Factor Va as a
platelet activation source, may result in
antibodies to Factors V and VI, with potentially life threatening coagulopathies
resulting.5 Other concerns with bovine
thrombin include prion disease, although
none are reported in the literature. The
authors have neither seen nor heard of
any infections occurring with the percutaneous use of PRP or biocellular therapeutic grafts.
Regarding the question of carcinogenesis, growth factors act on cell surface receptors only, do not enter the cell, and do
not cause DNA mutation. There is no
plausible mechanism by which growth factors would result in neoplastic development, and there have been no reports of
this in the literature.3,21 Furthermore,
Scott and Pawson showed that growth factors (PGF) activate normal, rather than
abnormal, gene expression.23
Typical Treatment Regimen With PRP
Consent
• Average series of injections is two to
three at four- to six-week intervals
• Different sites or areas of treatment
may expand or contract with further
treatment
• You must functionally retrain the
kinetic chain once the tissue has
undergone some degree of healing
Risks
• 1:50,000 chance of introducing
infection with injection procedure
• Allergy to local anesthetic(s)
• Syncope with pain/blood at the time
of injection
• Injury occurence with numbness or
pain following procedure. i.e.
falling, ankle sprain with inversion,
etc.
• Though extremely rare, pain or
function may worsen
• Puncture of tissue outside of intended graft site. i.e. vascular, neural,
lung, or other tissue placements
Technique for Myotendinous or TenoOsseous Sites
• Alcohol or Betadyne prep (we prefer
Betadyne gel when using an ultrasound probe for ‘live’ injection guidance)
• +/- Ethyl Chloride spray
• Inject PRP with approximately 1cc
PRP per cm3 of tissue/interface
• Important to touch bone and ‘pepper’ the area of teno-osseous junction to stimulate the greatest number of fibroblast colonies
• For myotendinous sites use ultrasound to ensure layered treatment
throughout the tendon
• Sterile band-aid applied post injection
• Kinesiotape to protect motion if
needed
Technique For Intra-Osseous Sites
• Alcohol or Betadyne prep (we prefer
Betadyne gel when using an ultrasound probe for ‘live’ injection guidance)
• +/- Ethyl Chloride spray
• Local anesthetic either mixed with
the PRP graft or to sites of tenderness to ‘road test’ the area prior to
using the graft. This ensures that the
PRP matrix graft is placed in the
proper areas.
• Aspirate degenerative joint fluid
prior to PRP matrix graft placement
• Gel the PRP or utilize other stabilizing matrix for intra-articular sites.
Ligaments, tendons, and inherent
matrix sites do not require gel in the
authors’ experience
• 8-10cc PRP matrix graft is the typical amount used for a knee or shoulder joint in our clinic
• “Treat regionally, not locally”(D.
Crane, MD; e.g. treat all of the capsule that is tender along with tendinous and ligamentous sites of tenderness in addition to the intraarticular capsule)
It should be noted that Kevy and Jacobson have evaluated the mixture of common
local anesthetics with PRP and find no significant platelet activation or diminution
of graft growth factor functions.7,24
Tendonosis and the Use of PRP
Anitua showed—from in vitro studies of
collagen and tendon—that autologous
preparations rich in growth factors promote proliferation and induce VEGF and
HGF production by human tendon cells
in culture.25 Mishra performed an in vitro
study which determined the effect of a
platelet concentrate medium on the proliferation of human skin fibroblasts—the
cells responsible for deposition of collagen. Buffered PRP was shown to augment
human fibroblast proliferation when compared to control.26
Schnabel evaluated gene expression
patterns, DNA, and collagen content of
equine flexor digitorum tendons cultured
in a media consisting of PRP and other
blood products. PRP at 100% concentration stimulated the greatest number of
collagen type I, collagen type III, and cartilage oligomeric protein (COMP) molecule genes without increasing expression
of the pro-inflammatory matrix metalloproteinases. ELISA detected higher levels of PDGF and TGF-B in the PRP
group.27
Hesham El-Sharkawy et al.10 measured
platelet derived growth factor (PDGF)-AB,
could be responsible for the release of this
growth factor.10
Tissue culture studies performed by du
Toit et al. for use in dermal regeneration
confirmed the potent mitogenic stimulation of human fibroblasts, keratinocytes,
chondrocytes, neural tissue, and myoblasts.28
In Vivo Human Studies: Reviews and Case
Examples
Tendon and Ligament Use of PRP
Mishra evaluated 20 patients that failed
non-operative treatment for chronic epicondylar pain. These 20 patients were
randomized to a single PRP injection or
injection with bupivicaine. Mishra comments that the IRB would not allow a
blood draw from the control patients to
blind the study. All PRP patients had
lower pain and greater ROM than control
(bupivicaine). Eight weeks after the treatment, the platelet-rich plasma patients
“Eight weeks after the treatment, the platelet-rich plasma patients
noted 60% improvement in their visual analog pain scores versus
16% improvement in control patients... At 6 months, the patients
treated with platelet-rich plasma noted 81% improvement in their
visual analog pain scores...”
PDGF-BB, transforming growth factor-b1,
insulin-like growth factor-I, fibroblast
growth factor-basic (FGF-b), epidermal
growth factor (EGF), vascular endothelial
growth factor, interleukin-12 (p40/70)
and, regulated on activation, normal Tcell expressed and secreted (RANTES) levels by enzyme-linked immunosorbent
assay. Cytokine, chemokine, and LXA4
levels, as well as monocyte chemotactic migration, were analyzed. PRP led to significantly increased levels of growth factors
and significantly suppressed inflammation by promoting secretion of LXA4.
These growth factors stimulated the
proliferation of fibroblasts and periodontal ligament cells, as well as extracellular
matrix formation, and promoted collagen
and total protein synthesis while stimulating the synthesis of hyaluronate from gingival fibroblasts. IGF-I levels in PRP in
this study were not significantly different
from the cyclolignan picropodophyllin
(PPP), suggesting that other cell types
noted 60% improvement in their visual
analog pain scores versus 16% improvement in control patients. Sixty percent
(three of five) of the control subjects withdrew or sought other treatments after the
8-week period, preventing further direct
analysis. Therefore, only the patients
treated with platelet-rich plasma were
available for continued evaluation. At six
months, the patients treated with plateletrich plasma noted 81% improvement in
their visual analog pain scores (P=.0001).
At final follow-up (mean, 25.6 months;
range, 12-38 months), the platelet-rich
plasma patients reported 93% reduction
in pain compared with before treatment
(P<.0001). Of importance, no PRP-treated patient was worse after treatment, and
there were no complications reported in
this study.29
Barrett et al. demonstrated, in a series
of nine plantar fascia patients, that PRP—
with ultrasound guidance—could be safely injected into the medial and central
5
FIGURE 4. Diagnostic ultrasound of the left
achilles (proximal and distal) showing a large
hypoechoic noninsertional tendonosis with surrounding fibrosis.
FIGURE 5. Diagnostic ultrasound of the left Achilles
taken 1.5 months after initial PRP matrix grafting showing improved collagen organization, lessened fibrosis, and improved capillary blood flow.
bands of the most affected plantar fascia
with promising results. Seven out of nine
patients had complete resolution of their
plantar fascial pain at one year and all the
patients in the study had improvement
that was noted on diagnostic ultrasound.
One of the patients was considered a failure because of a subsequent steroid injection even though all pain had resolved.30
Scarpone reported on a prospective
study carried out in 14 patients with shoulder pain. The patients all had rotator cuff
tears with no significant AC joint thickness
with impingement and no other significant symptomatic pathology such as labral
tears, glenohumeral arthrosis, or gross instability. All of the patients failed non-operative treatments such as NSAIDs, physical therapy, and corticosteroid injections
and all were considering surgical options.
Of the 14 patients, 12 had statistically significant improvements in their pain scale
and their strength and endurance at eight
weeks. Of the 12 patients, six had radiographic evidence of healing of their
tendinopathy on MRI at eight weeks. Of
the four patients who were considering
surgery because of persistent pain, only
two went on to have rotator cuff surgery.
No significant complications were noted.31
Ventura et al. evaluated PRP in ACL repair. A total of 20 patients with anterior
cruciate ligament (ACL) injuries were
treated by quadrupled hamstring tendon
graft (QHTG)—with or without PRP gel
growth factor (GF) application. CT highlighted a significant difference (P<0.01)
between ACL density of the two groups.
CT densities of the ACL and posterior
cruciate ligament (PCL) were similar in
the GF-treated group. In the control
group, however, the intensity of the signal was heterogeneous and the new ACL
was not clearly identifiable with respect to
the PCL. A different density of the ACL
was also noted: in the GF-treated group
this density was uniform and the new ACL
was more structured, while in the control
group the ligament was less structured
and did not completely fill the femoral
and tibial tunnels. In the PRP treated
group, one patient had a synovitic reaction. On CT, the new ACL was increased
and hypertrophic and surrounded by a
soft-tissue reaction. MRI confirmed this
finding.32
Sanchez reported on a case-control
study of twelve athletes with complete
achilles rupture. All twelve had open
achilles repair; six had PRGF. The treatment group had no wound complications
and experienced earlier functional
restoration: ROM (7 vs. 11 wks), jogging
(11 vs. 18 wks), and training (14 vs. 21
6
FIGURE 6. U/S pictures (3) proximal and distal chronic tendonopathy with scarring and
fibrosis of the VMO at the myotendinous
junction and insertion.
wks). The authors of this study measured
IGF-1, TGF-B1, PDGF-AB, EDF, VEGF,
and HGF and noted that the number of
platelets held direct correlation to the
level of growth factors.33
Case Example: Chronic Tendonopathy
A 63-year-old male ironman distance
triathlete presented with a history of left
achilles pain longer than three months.
The patient had no relief with physical
therapy or ultrasound (U/S) therapy for
a six week duration. The patient was diagnosed by MRI with stress fracture of the
fibula with no discrete cortical line or
fracture in addition to an achilles tendonopathy. Diagnostic U/S in our office
showed an 8cm segment of tendon collagen change consistent with a tendonopathy with associated peritenon fibrosis (see
Figure 4).
FIGURE 9. X-ray of left hip following PRP matrix graft series. X-ray shows subtle smoothing
of the irregular femoral head surface.
FIGURE 8. X-ray of pelvis and affected left hip
pre-treatment with PRP matrix graft.
FIGURE 7. U/S pictures (2) post injection
with PRP and gel matrix at the myotendinous junction and the insertion.
The patient undergoes three separate
series of PRP at four-week intervals to the
achilles tendon and fibula along with the
peroneal tendon sheath at the myotendinous junction. Subsequent ultrasounds
show improved fibrosis and less scarring
along with collagen pattern reorganization consistent with improved vascularity
and tendon structure (see Figure 5).
The patient has greater than 90% pain
reduction after the three PRP matrix
grafts and returns to ironman distance
racing after the three months of restricted training. Supportive compression
sleeves are utilized for three months to
allow for load distribution until strength
in the peroneal muscles and achilles is
90% of the unaffected right side.
Muscle Strain and the Use of PRP
Sanchez reported a 20 patient prospective muscle injury pilot study with sixmonth follow-up. Ultrasound guided injection of PRP within the injured muscle
enhanced healing (echo-graphic images)
and functional capacities 50% faster than
the control group.34
Case Example: Quadriceps VMO Muscle
Strain
A 56-year-old male presented with right
thigh pain occurring for approximately
one year. The pain is worse on the bike
and, in fact, is more prevalent when seated and pushing large gears or uphill
climbing. The patient has no significant
pain with running. The patient is an ironman distance triathlete and remembers
no injury of significance one year ago at
onset. Ultrasound shows a vastus medialis
injury/strain pattern with associated fiber
tearing and fibrosis. This is near the VMO
myotendinous junction at the right knee
(see Figure 6). No evidence of knee
pathology is noted on physical exam or
on ultrasound. Palpable tenderness exists
at the strain site on the medial thigh. Pain
is also reproduced on eccentric loading of
the VMO muscle group. No improvement
had been obtained previously with three
weeks of NSAID use, physical therapy, or
myofascial therapy work.
The patient undergoes a single injection of PRP (4cc) along with 1cc of injectable collagen for matrix stabilization
at two discrete sites in the VMO muscle
with ultrasound guidance (see Figure 7).
The patient’s pain after one month is
more than 80% resolved and the patient
has no pain on the bike or with activity as
previously noted. Resumption of training
occurred one week following injection with
swimming, running, and protected cycling.
Articular Cartilage and the Intra-Articular
Use of PRP
Everts, Devilee, et al. reported that autologous platelet gel and fibrin sealant enhance the efficacy of total knee arthroplasty by improved range of motion, decreased
length of stay, and a reduced incidence of
arthrofibrosis. Everts’ team also investigated whether the use of autologous derived
platelet gel and fibrin sealant would reduce
postoperative blood loss, decrease the impaired range of motion, and reduce the incidence of arthrofibrosis. Study group patients (n=85) were treated with the application of autologous platelet gel and fibrin sealant at the end of surgery. Eighty patients were operated without the use of
platelet gel and fibrin sealant and served
as the control group. During a five-month
postoperative period, patients were followed to observe the incidence of arthrofibrosis. In patients in the treatment group,
the hemoglobin concentration in blood
decreased significantly less when compared to the control group. They also
showed a superior postoperative range of
motion when compared to those of the
control group (P<0.001). The incidence of
arthrofibrosis and subsequent forced manipulation was significantly less (P<0.001)
in patients managed with platelet gel and
fibrin sealant.35
Case Example: Severe Hip Osteoarthritis
With a History of Congenital Hip Dysplasia
A 56-year-old female presented with increasing left hip pain greater than one
year duration. The patient has a history of
bilateral hip dislocations at birth (birth
country Poland — no x-rays available) with
evidence of shallow acetabular deformity
noted on x-ray (see Figure 8).
The patient is active in dance and is of
normal weight and BMI. Some relief is
obtained with NSAID therapy but pain is
now affecting sleep and is interfering with
activities of daily living and her dance regimen. The patient undergoes one PRP injection to the left hip using an anterior
approach. 8cc PRP is placed with ultrasound guidance as noted (see Figure 9).
7
FIGURE 10. Initial MRI of the LS spine shows
right sided pedicle stress reaction at vertebral
level L5.
After 3 months, the patient reports 75%
pain improvement and some improvement in ROM is also reported. The night
pain has resolved and the patient’s pain
is controlled with acetaminophen. She is
able to resume dance and activities for fitness and health.
Bone and Periosteal Use Of PRP
Gandhi et al. observed normalized cellular proliferation and chondrogenesis with
an improved mechanical strength when
PRP was injected percutaneously in a diabetic experimental femur fracture model.36
Sanchez et al. utilized PRP after reattachment of a large (2 cm) loose chondral
body in its crater in the medial femoral
condyle. Autologous plasma (PRP) was injected into the area between the crater
and the fixed fragment. They state that
complete articular cartilage healing was
considerably accelerated, and the functional outcome was excellent, allowing a
rapid resumption of symptom-free athletic activity.37
PRP has been used successfully in maxillofacial surgery in several studies including a randomized trial of 88 patients with
mandibular defects treated with cancellous cellular marrow grafts with, or without, PRP. Grafts with PRP showed twice
the radiographic maturity at six months
follow up.2
8
FIGURE 11. MRI repeat LS spine (approx. 1
year following the initial MRI) with evidence
of new left pars defect at L5 with evident partial healing of right sided stress fracture.
Another case report describes a fiftyyear old woman with nonunion of
humerus who had undergone two unsuccessful operations. Union was obtained
by the use of autologous platelet-rich gel
(PRG). At the 8th week, over 75% of the
circumference of the bone at the defect
site had resolved and, during later visits, remodeling of the union was observed on X-ray films and DEXA examinations. Maximum healing was reached
at the 18th week. Twelve months after
PRG injection, the intramedullary nail
that had previously been placed was removed.38
Case Example: Bilateral Pars Interarticularis Stress Fractures (Spondylolysis)
A 14-year-old softball player presented
with a history of developing back pain
over a period of six weeks, made worse
following a minor motor vehicle crash
four weeks prior to visit. The patient had
initial pain and localized tenderness on
the right low back L4-5 area with a positive stork test. X-ray and MRI confirm
spondylolysis (see Figure 10).
The patient undergoes extensive physical therapy for approximately 8 months
with subsequent relief. The patient then
returns to sport specific activity but redevelops pain. After appropriate discussion
FIGURE 12. Repeat MRI following four rightsided and three left-sided PRP matrix grafts
with evident interval healing.
of the benefits and risks, a PRP matrix
graft is placed on the right L5-S1 facet
joint and the L5 pars with ultrasound
guidance. On return to activity, the patient notes the absence of pain on the
right pars or low back area. The patient
is allowed to slowly return to activity. Two
months following the initial PRP graft,
the patient develops pain in the opposite,
left lumbar area after repeated throwing
drills. A repeat MRI shows a left sided
spondylosis. No listhesis is appreciated.
Evidence of healing is noted on the right
pars stress fracture to a small degree (see
Figure 11).
A PRP matrix graft—with a total 8cc
PRP at a six-fold concentration and
mixed with 2 cc 50:50 lidocaine 1% with
marcaine 0.5%—is then placed an additional X3 on the right and X3 on the left,
with approximately 5cc placed at the lev-
els of the L5 pars as well as the accompanying facet joints. The patient is started
on physical therapy at two weeks into the
graft injection series with progression at
6 weeks to pilates therapy and then sport
specific activity with heavy focus on the
mechanics of core stabilization and kinetic chain reintegration. A repeat MRI is
obtained two months following PRP (see
Figure 12).
Figure 12 shows interval slight healing
of the fracture sites. The patient has not
developed any reoccurrence of pain and
is back to softball activities with no bracing. No tenderness remains at the prior
fracture sites on physical exam.
In Vivo Studies: Skin Healing, Range of
Motion, and Pain With the Use of PRP
A prospective, single-blind pilot study
comprising 80 full-thickness skin punch
wounds (4mm diameter) was conducted
on the thighs of eight healthy volunteers.
With each subject serving as his or her
own control (five punch sites per leg), PRP
was applied topically on one thigh, while
an antibiotic ointment and/or a semi-occlusive dressing was applied on the other
thigh. On day 17, the percentage of closure was 81.1% for the PRP-treated sites
and 57.2% for the control sites. Also, the
PRP wound closure velocities were significantly faster than those of the controls
(P=.001). When the platelet count in the
gel was more than six times the baseline
intravascular platelet count in some subjects, epithelialization and granulation
formation appeared three days earlier in
the PRP-treated group.39
Everts et al. noted improved wound
healing when platelet leukocyte gel was
applied during wound closure after total
knee arthroplasty.5
In a study examining PRP gel for diabetic foot ulcers, Driver et al. noted that
13 of 19 patients in the study group
(68.4%) had complete healing, compared
with only 9 of 21 (42.1%) of the control
group (saline gel). This study was a
prospective, randomized, controlled trial
with both groups receiving a blood draw
for blinding purposes. The treating
providers and patients were blinded to
the gel applied. It should be noted that
no treatment serious adverse events were
reported and bovine thrombin used for
PRP gel did not cause any Factor V inhibition.40
In another study from Everts et al.,
platelet leukocyte gel (PLG) was injected
in the subacromial space during wound
closure in patients who underwent an
open subacromial decompression.41 In
the PLG-treated patients, a decrease in
the VAS pain score was observed
(P<0.001) compared to the non-treated
patients. Consequently, the use of pain
medication was significantly less
(P<0.001) in PLG-treated patients. Furthermore, treated patients demonstrated
a significantly improved range of motion
earlier after surgery with a high shoulder
functional index.
A significant reduction in pain was also
observed after PRP use by Fanning et al.
after applications in gynecologic surgery42; Gardner and co-workers following
total knee replacement surgery43; and
Crovetti and associates in patients with
chronic wounds.44
Conclusion
PRP matrix grafts along with other biologic grafting techniques are becoming
more prevalent in the treatment paradigms of musculoskeletal medicine.
These PRP matrix grafts provide effective, safe, relatively low-cost treatment options to patients who have the time and
wherewithal to allow collagen synthesis
and maturation at the graft site. PRP matrix grafts appear to restore tissue homeostasis and biotensegrity of collagen.
Other pain inhibiting effects are also present in PRP matrix grafts which allow earlier resumption of pain free activity. It is
the authors’ experiences that these grafts,
along with other regenerative grafting
options, are at times the only viable treatment option for a select group of patients
with degenerative myofascial tissue injuries. The authors recommend appropriate first line therapies such as relative rest,
appropriate bracing and kinesiotaping,
evaluation of kinetic chain mechanics,
and physical therapy—with or without eccentric loading protocols—prior to the
utilization of these PRP matrix grafting
protocols.
Reduction in pain after PRP applications has been observed by several authors. However, an explanation of this
phenomena has not always been given.
The authors believe that serotonin released from activated platelets might be
responsible for decreased pain, as described by Everts41 and Fanning.42 Except
for the growth factors in the Alpha-granules, large amounts of serotonin45 are contained within the dense platelet granules.
Since platelet counts of the PRP are generally almost six-fold higher when compared to whole blood levels, it stands to
reason that serotonin levels are therefore
also significantly increased at the wound
site. This phenomena has been explained
in detail by Sprott et al.46 who reported on
pain reduction following acupuncture
and measured a decrease in serotonin
concentration in platelets from these patients and an increase in serotonin levels
in plasma—suggesting normalization of
plasma serotonin levels due to the mobilization of platelet serotonin.
Other grafting tools such as the use of
autologous bone marrow aspirate stem
cells (BMAC) with PRP matrices have not
been explored in this article but may be
found in further detail by the authors.
These stem cell/growth factor grafts are
being utilized for severe degenerative
states with associated tissue hypoxemia.
Hence, PRP and other regenerative biocellur therapeutic matrices deserve further study to determine their effects in animal and human models. n
David M. Crane, MD practices Sports Medicine in Chesterfield and Saint Louis, Missouri. He is Board Certified in Emergency
Medicine and Sports Medicine. Dr. Crane is
the founder and Director of the Crane Clinic
Sports Medicine. Dr. Crane utilizes the most
up to date therapies to assist the healing process
in his patients, including Regenerative Injection Therapy (RIT) which uses the patient's
own growth factors, or bioactive proteins, to encourage the in-growth of normal collagen and
remodeling of injured tissue. He may be contacted at: Crane Clinic Sports Medicine, 219
Chesterfield Towne Centre, Chesterfield, MO
63005, or by email through his website at
www.craneclinic.com.
Peter A.M. Everts, PhD heads the Department of Peri-Operative Blood Management at
Catharina Hospital at Eindhoven, The
Netherlands. He is currently the chairman of
Foundation FERET which is the research
leader in the PRP medical field. Dr. Everts has
done pioneering work involving autologous
platelet rich plasma in both animal and human
trials and wrote the 2007 thesis titled: Autologous Platelet-Leukocyte-Enriched Gel:
Basics and Efficacy, which describes a novel
method to support soft tissue and bone healing
(ISBN 10:90-8590-016-6, ISBN 13:978-908590-016-0). Dr. Everts may be contacted via
email at everts@elive.nl.
9
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Platelet Rich Plasma (PRP) Matrix Grafts

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